Gfp 1020

Manufactured by Thermo Fisher Scientific

The GFP-1020 is a laboratory instrument designed for the detection and analysis of green fluorescent protein (GFP) in samples. The device utilizes fluorescence detection technology to measure the presence and quantity of GFP, a commonly used reporter protein in biological research. The core function of the GFP-1020 is to provide accurate and reliable GFP quantification capabilities for scientific applications.

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9 protocols using gfp 1020

Immunohistochemistry of Cutaneous Nerves

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Mice were euthanized with CO₂, transcardially perfused with PBS/4% paraformaldehyde (PFA). Back hairy skin tissue (de haired with Nair) and footpad of hind paw were dissected and post-fixed with 4% PFA for 2 hours at 4°C, and cryo-protected in 30% sucrose in 1XPBS overnight. Frozen sections of 40μm were cut using a Leica CM1950 cryostat. Immunostaining of sections, and whole mount hairy skin were performed as described previously [24] (link), [25] (link). Antibodies used are as follows: rat anti-cytokeratin 8 (DSHB, TROMA1, 1∶20), goat anti-TrkC (R&D, AF1401, 1∶1000), chicken anti-GFP (Aves, GFP-1020, 1∶1000), rabbit anti-GFP (Invitrogen, A-11122, 1∶2000), chicken anti-NFH (Aves, NF-H, 1∶1000), rabbit anti-NFH (Sigma, N4142, 1∶1000), and Alexa Fluorescent conjugated Goat or Donkey secondary antibodies (Invitrogen or Jackson IR, 1∶500-1000).

Niu J., Vysochan A, & Luo W. (2014). Dual Innervation of Neonatal Merkel Cells in Mouse Touch Domes. PLoS ONE, 9(3), e92027.

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Immunohistochemical Labeling of Spinal Cord

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Immunohistochemistry was performed on fixed frozen sections as previously described (Niu et al., 2013 (link)) using chicken anti-GFP (Aves, GFP-1020, 1:1000), rabbit anti-GFP (Invitrogen, A-11122, 1:2000), chicken anti-NFH (Aves, NF-H, 1:1000), rabbit anti-NF200 (Sigma, N4142, 1:1000), mouse anti-NFM (Developmental Studies Hybridoma Bank (DSHB), 2H3, 1:100), mouse anti-NCAM (DSHB, 5A5, 1:100), rabbit anti-ZIC2 (generated in the Herrera lab), goat anti-PGD2 synthase (Santa Cruz, sc-14825, 1:200), mouse anti-NESTIN (BD Pharmingen, 1:100), mouse anti Isl1 (DSHB, 39.4D5, 1:5), mouse anti-Lhx1/5 (Lim1/5) (DSHB, 4F2, 1:5), rabbit anti-Pax2 (Proteintech, 21385-1ap, 1:200), guinea pig anti-Lmx1b (T. Perlmann, Karolinska Institute, Stockholm, Sweden, 1:2000) and Alexa Fluorescent conjugated Goat or Donkey secondary antibodies (Invitrogen or Jackson IR, 1:500-1000). Semi-thin spinal cord sections were performed as previously described (Niu et al., 2013 (link)).

Kridsada K., Niu J., Wang Z., Haldipur P., Ding L., Li J.J., Lindgren A.G., Herrera E., Thomas G.M., Chizhikov V.V., Millen K.J, & Luo W. (2018). Roof Plate-Derived Dorsal Midline Radial Glial-like Cells Promote Longitudinal Ascending Axon Growth of Rapidly Adapting Mechanoreceptors during Development. Cell reports, 23(10), 2928-2941.

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Immunofluorescence Staining of Cytoskeletal Proteins

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For Structured Illumination Microscopy (SIM) and A1 confocal imaging, cells were washed with pre-warmed phosphate-buffered saline (PBS) before being fixed in 4% paraformaldehyde (Electron Microscopy Sciences #15710) in PBS for 15 mins at 37°C. After fixation, cells were rinsed three times in PBS and permeabilized with 0.1% Triton X-100 in PBS for 15 mins at 37°C. If cells were stained with anti-Fascin, cells were fixed and permeabilized with ice cold methanol on ice for 15 mins instead of 4% paraformaldehyde. After permeabilization, cells were rinsed three times in PBS and blocked with 5% BSA (Research Products International #9048-46-8) in PBS for 1 hr at 37°C. Immunostaining was performed using the primary antibodies: anti-VASP (1:50; Cell Signaling Technologies #3132S), anti-Fascin (1:100; Agilent Technologies #M356701-8), anti-GFP (1:200; Aves Labs #GFP-1020), anti-mCherry (1:500; Invitrogen #M11217), diluted in 1% BSA in PBS at 37°C for 1 hr. After incubation with primary antibody, coverslips were rinsed three times in PBS and incubated for 1 hr with Alexa Fluor 568-phalloidin or Alexa Fluor 647-phalloidin (1:200; Invitrogen #A12380 and #A22287) at room temperature. Coverslips were then washed five times in PBS and mounted on glass slides in ProLong Gold (Invitrogen #P36930).

Fitz G.N., Weck M.L., Bodnya C., Perkins O.L, & Tyska M.J. (2023). Protrusion growth driven by myosin-generated force. Developmental cell, 58(1), 18-33.e6.

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Immunostaining of Mouse Neural Tissue

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Procedures were conducted as previously described [5 (link)]. Briefly, mice (>6 weeks old) used for immunostaining were anesthetized with ketamine/xylazine/acepromazine and transcardially perfused with 4% PFA/PBS, and dissected tissue (brain, spinal cord, or DRGs) was post-fixed for 2 hr-overnight in 4% PFA/PBS at 4° C. Tissue used for immunostaining was then sectioned (100 μm) using a T1200S vibratome (Leica Microsystems, Nussloch, Germany). Immunostaining of sections was performed as described previously [5 (link),6 (link)]. The following primary antibodies were used: chicken anti-GFAP (Aves Labs Cat# GFAP, RRID:AB_2313547), chicken anti-GFP (Aves Labs Cat# GFP-1020, RRID:AB_10000240), rabbit anti-GFP (Thermo Fisher Scientific Cat# A-11122, RRID:AB_221569), mouse anti-NeuN (Millipore Cat# MAB377, RRID:AB_2298772).

Prabhakar A., Vujovic D., Cui L., Olson W, & Luo W. (2019). Leaky expression of channelrhodopsin-2 (ChR2) in Ai32 mouse lines. PLoS ONE, 14(3), e0213326.

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Antibody Staining of Neural Markers

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Anti-POGZ was prepared as described^{26 (link)}; chicken anti-GFP (Aves Laboratories GFP‐1020 1:1000); rabbit anti-Ki67 (Thermo Scientific RB-1510 1:1000); rabbit anti-Pax6 (BioLegend PRB 278P 1:500); rabbit anti-TBR2 (ab23345 1:250); rabbit anti-phospho-histone H3 (Cell Signaling Technology 9701 1:400) Alexa Fluor® 488-labeled IgG was used as secondary antibody (ab150077 1:200).

Suliman-Lavie R., Title B., Cohen Y., Hamada N., Tal M., Tal N., Monderer-Rothkoff G., Gudmundsdottir B., Gudmundsson K.O., Keller J.R., Huang G.J., Nagata K.I., Yarom Y, & Shifman S. (2020). Pogz deficiency leads to transcription dysregulation and impaired cerebellar activity underlying autism-like behavior in mice. Nature Communications, 11, 5836.

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Immunohistochemical Profiling of Murine Spinal Cord

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Young adult mice were deeply anaesthetized and fixed by perfusion with 4% paraformaldehyde. The spinal cord was dissected and post-fixed in 4% paraformaldehyde overnight at 4°C then rinsed thoroughly with 1X phosphate buffer saline (PBS). Transverse sections 60 µm thick were cut with a vibrating microtome from mid-lumbar spinal cord segments (L2 or L3) and processed free-floating for immunocytochemistry. Sections were washed in 50% ethanol for 30 min, rinsed 4 × 5 min in PBS containing 0.3% Triton X-100 (Sigma, St. Louis MI), and incubated in primary antibodies for three nights at 4°C. These were revealed with species-specific secondary antibodies raised in donkey and conjugated to Alexa fluor 488, Alexa fluor 555, or Alexa fluor 647 (Life Technologies, Carlsbad, CA) diluted 1:500 in and incubated overnight at 4°C. Sections were scanned with a confocal microscope (Nikon AR1) through a 20x or 60x oil-immersion. Scans were analyzed with NeNIS Elements Software (Nikon, Melville NY). Primary antibodies used were chicken anti-green fluorescent protein (Aves Labs, Tigard OR, GFP-1020, 1:2000), rabbit-anti Pax2 (ThermoFisher Scientific, Waltham MA, 71–6000, 1:1000), mouse anti-NeuN (Millipore, Billerica MA, MAB377, 1:1000), and rabbit-anti Nos1 (ThermoFisher Scientific, 61–7000, 1:500).

Hachisuka J., Baumbauer K.M., Omori Y., Snyder L.M., Koerber H.R, & Ross S.E. (2016). Semi-intact ex vivo approach to investigate spinal somatosensory circuits. eLife, 5, e22866.

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Immunohistochemical Analysis of Protein Expression

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Mice were killed by intraperitoneal injection injection of pentobarbital (150 mg/kg; Nembutal, Abbott Laboratories) and perfused with 4% paraformaldehyde. Tissue sections were incubated in 5% bovine serum albumin and 0.3% Triton X-100 (Sigma) to block nonspecific binding and then incubated with primary antibodies overnight at 4℃, followed by incubation with secondary antibodies at room temperature. Antibodies include anti-anti-GFP (Aves labs, GFP-1020), anti-HDAC1 (PA1-860, Thermo Scientific), anti-HDAC2 (H2663, Sigma), anti-Phospho-p53 Ser15 (9284, Cell Signaling), and anti-PUMA (7467, Cell Signaling). After washing with phosphate-buffered saline, sections were incubated with secondary antibodies coupled to Alexa 488 (1:250, Invitrogen) or Cy3 (1:250, Jackson Laboratories) for 1 hr at room temperature, washed in phosphate-buffered saline, and mounted with IMMU-MOUNT (Thermo Scientific). Staining was observed using a fluorescence microscope (Axioplan2 Imaging, Carl Zeiss). Images were digitized with a Powershot G5 camera (Canon) and acquired with Axio Vision 4.5 software (Carl Zeiss). Images were further processed (levels adjusted) using Photoshop CS5 software (Adobe).

Lebrun-Julien F, & Suter U. (2015). Combined HDAC1 and HDAC2 Depletion Promotes Retinal Ganglion Cell Survival After Injury Through Reduction of p53 Target Gene Expression. ASN NEURO, 7(3), 1759091415593066.

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Quantifying NFIX+ and GFP+ Cells in BNSTp

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Brains were dissected from perfused P14 Esr1^Cre/+;Sun1–GFP^lx/+ animals and cryosectioned at 40 μm before immunostaining with primary antibodies to GFP (1:1,000, Aves GFP-1020) and Nfix (1:1,000, Thermo Fisher PA5-30897), and secondary antibodies against chicken (1:300, Jackson Immuno 703-545-155) and rabbit (1:800, Jackson Immuno 711-165-152), as previously described^{16 (link)}. A Zeiss Axioimager M2 System equipped with MBF Neurolucida Software was used to take 20× wide-field image stacks spanning the BNSTp (five sections, both sides). The number of Nfix⁺, GFP⁺ and Nfix⁺GFP⁺ cells was quantified using Fiji/ImageJ from the centre three optical slices by an investigator blinded to condition.

Gegenhuber B., Wu M.V., Bronstein R, & Tollkuhn J. (2022). Gene regulation by gonadal hormone receptors underlies brain sex differences. Nature, 606(7912), 153-159.

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Multimodal Analysis of Regenerating Zebrafish Fins

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Dissected 32 dpf shha:GFP caudal fins were fixed in 4% PFA/PBS overnight at 4°C. After extensive PBS washing, fins were decalcified for 4 days in 0.5M EDTA, pH 8.0 with daily solution changes. Fins then were dehydrated in an ethanol series and tissue cleared with xylenes prior to longitudinal embedding in paraffin wax. 7 µm sections were cut on a Leica RM255 microtome. Antigen retrieval was performed on rehydrated sections using 1 mM EDTA + 0.1% Tween-20 for 5 minutes in a pressure cooker. Following PBS washes, sections were blocked in 1x PBS, 10% nonfat dry milk, 2% normal goat serum, and 4% fetal bovine serum for a minimum of 1 hour. Sections were incubated overnight at 4°C with primary antibodies in blocking solution. Primary antibodies were: anti-GFP (1:3000; AVES, GFP-1020), anti-Tp63 (1:100; Thermo Fisher, PA5-36039), anti-Laminin (1:40; Sigma, L9393), and anti-Zns5 (1:5, ZIRC).
Sections were washed in PBS containing 500 mM NaCl + 0.1% Tween-20. Alexa Fluor conjugated secondary antibodies (Thermo Fisher) were diluted 1:1000 in blocking buffer and incubated for 1 hour at room temperature protected from light. Sections were washed, nuclei stained with Hoechst, and mounted with SlowFade Gold Antifade (Thermo Fisher). Images were acquired on a Zeiss LSM 880 laser scanning confocal microscope and images processed with Fiji-ImageJ, Imaris, and Adobe Photoshop.

Braunstein J.A., Robbins A.E., Stewart S., & Stankunas K. (2020). Basal epidermis collective migration and local Sonic hedgehog signaling promote skeletal branching morphogenesis in zebrafish fins.

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